Mutant glucose dehydrogenase

ABSTRACT

Substrate specificity for glucose of a glucose dehydrogenase having the amino acid sequence of SEQ ID NO: 13 is improved by substituting another amino acid residue for the amino acid residue at position 472 and/or 475.

RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. §371 of InternationalApplication PCT/JP2005/007687, filed Apr. 22, 2005, which was publishedin a language other than English, which claims priority of JPApplication No. 2004-128165, filed Apr. 23, 2004.

TECHNICAL FIELD

The present invention relates to a mutant glucose dehydrogenase showingimproved substrate specificity. The mutant glucose dehydrogenase of thepresent invention can be suitably used for glucose sensors, glucoseassay kits and so forth, and is useful in the fields of biochemistry,clinical medicine, and so forth.

BACKGROUND ART

In recent years, a variety of enzymes are used as biosensor elements.Glucose oxidases (GODs) have already been practically used as sensorelements for measuring blood glucose levels for the purpose of diagnosisof diabetes. However, GODs suffer from a problem that they are affectedby dissolved oxygen in samples. Therefore, glucose dehydrogenases(GDHs), which are not affected by dissolved oxygen in samples, aredrawing attentions as alternatives of GODs.

As GDHs, one requiring NAD(P)⁺ as a coenzyme (E.C.1.1.1.47), onerequiring pyroloquinoline quinone (PQQ) as a coenzyme (PQQGDH;E.C.1.1.99.17) etc. have been reported. GDH requiring NAD(P)⁺ as acoenzyme suffers from a problem as a sensor element that NAD(P)⁺ needsto be added to the assay system. On the other hand, it is unnecessaryfor coenzyme-binding type GDHs such as PQQGDH to add a coenzyme to theassay system.

Further, sensor elements are desired to exhibit a stability that thefunction as a sensor is not lost even when they are continuously used orleft at room temperature.

Since enzymes derived from thermophilic bacteria which grow at hightemperature generally exhibit high thermostability, and high stabilityeven in long-term storage, continuous use and so forth, application ofthem as sensor elements is expected. However, although GDHs derived fromThermoplasma acidophilum and Sulfolobus solfataricus have been reportedas thermostable GDHs derived from thermophilic bacteria, both of themrequire NAD(P)⁺ as a coenzyme.

On the other hand, thermostable GDH produced by Burkholderia cepacia, amoderately thermophilic bacterium, is an FAD-binding type GDH, and theenzymological characteristics thereof such as optimum reactiontemperature, thermostability and substrate specificity have already beenelucidated (Patent document 1). This GDH usually exists as aheterooligomer consisting of a catalytic subunit (α-subunit) showinghigh heat resistance, an electron transfer subunit (β-subunit), which iscytochrome C, and γ-subunit of which function is unknown, and itsoptimum reaction temperature is 45° C. These subunits are dissociated bya heat treatment at a temperature higher than 50° C. to release theα-subunit monomer of which optimum reaction temperature is 75° C. Theα-subunit monomer is thermostable and exhibits 80% or more of residualactivity even after a heat treatment at 60° C. for 30 minutes. The genescoding for these subunits have also already been isolated (Patentdocuments 1 and 2).

However, coenzyme-binding type GDHs generally exhibit a broad substratespecificity, and also react with maltose, galactose and so forth inaddition to glucose. When they are applied as a glucose sensor formonitoring blood sugar levels of diabetic patients, and the diabeticpatients have such severe symptoms that peritoneal dialysis must beperformed, there is a risk that values higher than the true blood sugarlevels may be obtained, because a large amount of maltose is containedin the dialysate. GDH derived from Burkholderia cepacia also exhibitsreactivity to maltose and galactose in addition to glucose.

A technique of changing substrate specificity of GDH by introducing anamino acid substitution mutation is known. As such mutant GDHs, forexample, there are known PQQGDHs derived from E. coli (Patent documents3 and 4), Acinetobacter calcoaceticus (Gluconobacter calcoaceticus)(Patent document 5), and Acinetobacter baumannii (Patent documents 6 to8) requiring pyroloquinoline quinone as a coenzyme.

-   [Patent document 1] U.S. Patent Application No. 2004/0023330-   [Patent document 2] International Patent Publication WO03/091430-   [Patent document 3] Japanese Patent Laid-open (Kokai) No. 10-243786-   [Patent document 4] Japanese Patent Laid-open No. 2001-197888-   [Patent document 5] Japanese Patent Laid-open No. 2004-173538-   [Patent document 6] Japanese Patent Laid-open No. 2004-313172-   [Patent document 7] Japanese Patent Laid-open No. 2004-313180-   [Patent document 8] Japanese Patent Laid-open No. 2004-344145

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an FAD-binding type GDHshowing an improved substrate specificity to glucose.

The inventors of the present invention conducted various researches inorder to achieve the foregoing object. As a result, they found that bymodifying the amino acid sequence of the FAD-binding type GDH derivedfrom Burkholderia cepacia at a specific site, the reactivity thereof tosugars other than glucose could be decreased while maintaining thereactivity to glucose, and thus accomplished the present invention.

That is, the present invention provides the followings.

-   (1) A mutant glucose dehydrogenase exhibiting improved substrate    specificity to glucose, which is a protein having the amino acid    sequence of SEQ ID NO: 13 or an amino acid sequence of SEQ ID NO: 13    including substitution, deletion, insertion or addition of one or    more amino acid residues at position other than the positions listed    below and having a glucose dehydrogenase activity, and has any of    the amino acid substitution mutations listed below (numerals    represent a position in the amino acid sequence, the amino acid    residues represent an amino acid residue after substitution at the    position, and “+” means that two amino acid substitutions are    simultaneously included):-   (A) 472Arg, 472Asn, 472Asp, 472Cys, 472Glu, 472Gly, 472H is, 472Ile,    472Leu, 472Met, 472Phe, 472Pro, 472Ser, 472Trp, 472Tyr, 472Val,-   (B) 475Asp, 475Cys, 475Glu, 475Gly, 475H is, 475Met, 475Phe, 475Ser,    475Tyr, 475Val,-   (C) 472Arg+475(Asp, Glu, Gly, H is, Phe, Ser, Tyr), 472Asn+475(Asp,    Gly, H is, Phe, Ser, Tyr), 472Asp+475(H is, Phe, Ser, Val),    472Cys+475(Asp, Gly, H is, Phe, Ser), 472Glu+475(Asp, Glu, Gly, H    is, Phe, Ser, Tyr), 472Gly+475(Asp, Cys, Gly, Met, Phe, Ser, Tyr),    472His+475(Cys, Glu, H is, Met, Phe, Ser, Tyr), 472Ile+475(Asp, Cys,    Glu, Gly, H is, Met, Phe, Ser, Tyr), 472Leu+475(Asp, Gly, H is, Phe,    Ser, Tyr), 472Met+475(Asp, Gly, H is, Phe, Ser), 472Phe+475(Asp,    Glu, Gly, H is, Met, Phe, Ser, Tyr), 472Pro+475His 472Ser+475(Asp,    Glu, Gly, H is, Phe, Ser), 472Trp+475(H is, Phe, Ser),    472Tyr+475(Asp, His, Phe, Ser), 472Val+475(Asp, Glu, Gly, His, Phe,    Ser).-   (2) The aforementioned mutant glucose dehydrogenase, which has the    amino acid sequence of SEQ ID NO: 13 provided that it includes any    of the amino acid substitution mutations listed in the    aforementioned (A) to (C).-   (3) The aforementioned mutant glucose dehydrogenase, which has an    amino acid substitution mutation selected from the following    mutations:-   (D) 472Arg, 472Asn, 472Asp, 472Glu, 472Gly, 472Phe, 472Pro,-   (E) 475Asp, 475Cys, 475Glu, 475Gly, 475Met, 475Phe-   (F) 472Arg+475(Asp, Gly, His, Phe), 472Asn+475(Gly, His, Phe, Tyr),    472Asp+475(H is, Ser), 472Cys+475(Gly, His, Phe), 472Glu+475(Glu,    His, Phe, Tyr), 472Gly+475(Asp, Phe, Tyr), 472His+475(H is, Ser),    472Ile+475(Asp, Glu, Gly, His, Ser), 472Leu+475(Gly, His, Phe, Tyr),    472Met+475(Asp, Gly, His, Phe), 472Phe+475(Asp, Glu, Gly, His, Phe,    Ser, Tyr), 472Ser+475(Glu, Gly, His, Phe), 472Trp+475(H is, Phe),    472Tyr+475His, 472Val+475(Asp, Glu, Gly, His, Phe).-   (4) A glucose dehydrogenase, which is a protein having the amino    acid sequence of SEQ ID NO: 13 or an amino acid sequence of SEQ ID    NO: 13 including substitution, deletion, insertion or addition of    one or more amino acid residues at position other than the positions    listed below and having a glucose dehydrogenase activity, and    wherein:-   (i) at least either the arginine residue at position 472 or the    asparagine residue at position 475 in the amino acid sequence of SEQ    ID NO: 13 is replaced with another amino acid residue, and-   (ii) a ratio of specific activity for glucose and specific activity    for maltose ((reactivity to maltose/reactivity to glucose)×100) of    the glucose dehydrogenase introduced with the aforementioned    mutation is reduced by 10% or more compared with that of a glucose    dehydrogenase not introduced with the mutation.-   (5) A mutant glucose dehydrogenase complex comprising at least the    aforementioned mutant glucose dehydrogenase and an electron transfer    subunit.-   (6) A DNA coding for the aforementioned mutant glucose    dehydrogenase.-   (7) A microorganism having the aforementioned DNA and producing the    aforementioned mutant glucose dehydrogenase or the mutant glucose    dehydrogenase complex.-   (8) A glucose assay kit comprising the aforementioned mutant glucose    dehydrogenase, the mutant glucose dehydrogenase complex, or the    microorganism.-   (9) A glucose sensor comprising the aforementioned mutant glucose    dehydrogenase, the mutant glucose dehydrogenase complex, or the    microorganism.-   In the present specification, although the term “mutant GDH” refers    to a mutant α-subunit in the context of contrast with a mutant GDH    complex, a mutant α-subunit and a mutant GDH complex may also be    collectively referred to as “mutant GDH”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dehydrogenase activities of DH5α/pTrc99A/γ+α for glucoseand maltose as substrates. The rhombuses represent the activity forglucose, and the squares represents the activity for maltose (the sameshall apply in FIGS. 2 to 5).

FIG. 2 shows dehydrogenase activities of DH5α/pTrcγαAsn475Asp forglucose and maltose as substrates.

FIG. 3 shows dehydrogenase activities of DH5α/pTrc99Aγαβ for glucose andmaltose as substrates.

FIG. 4 shows dehydrogenase activities of DH5α/pTrcγαβAsn475Asp forglucose and maltose as substrates.

FIG. 5 shows dehydrogenase activities of DH5α/pTrcγαβAsn475Glu forglucose and maltose as substrates.

FIG. 6 shows sequences of PCR primers used for codon substitutions atpositions 472 and 475 in the GDH α-subunit.

FIG. 7 shows SV plots of mutant GDHs.

FIG. 8 shows SV plots of mutant GDHs.

FIG. 9 shows a structure of a glucose sensor.

FIG. 10 shows reagent parts of a glucose sensor.

FIG. 11 shows reactivity to glucose of a glucose sensor using a wildtype GDH.

FIG. 12 shows reactivity to glucose of a glucose sensor using472Glu475Tyr type GDH.

FIG. 13 shows reactivity to glucose of a glucose sensor using472Asp475His type GDH.

FIG. 14 shows reactivity to maltose of a glucose sensor using a wildtype GDH in the presence of glucose.

FIG. 15 shows reactivity to maltose of a glucose sensor using472Glu475Tyr type GDH in the presence of glucose.

FIG. 16 shows reactivity to maltose of a glucose sensor using472Asp475His type GDH in the presence of glucose.

FIG. 17 shows apparent blood sugar levels measured by using glucosesensors using a wild type GDH and a mutant GDH.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained in detail.

The mutant GDH of the present invention is produced by introducing aspecific mutation into a wild type GDH. Examples of the wild type GDHinclude GDHs produced by Burkholderia cepacia. Examples of the GDHsproduced by Burkholderia cepacia include GDHs produced by theBurkholderia cepacia KS1, JCM2800 and JCM2801 strains. The KS1 strainwas deposited at the independent administrative corporation, NationalInstitute of Advanced Industrial Science and Technology, InternationalPatent Organism Depositary (Tsukuba Central 6, 1-1, Higashi 1-Chome,Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Sep. 25, 2000 and given anaccession number FERM BP-7306. The JCM2800 and JCM2801 strains arestored at the independent administrative corporation, RIKEN, BioresourceCenter, Japan Collection of Microorganisms (JCM).

The nucleotide sequence of a chromosomal DNA fragment containing the GDHα-subunit gene and a part of the β-subunit gene of the KS1 strain isshown in SEQ ID NO: 4 (U.S. Patent Application No. 2004/0023330). Threeopen reading frames (ORF) exist in this nucleotide sequence, the secondand third ORFs from the 5′ end side code for the α-subunit (SEQ ID NO:13) and the β-subunit (SEQ ID NO: 14), respectively. Further, it isinferred that the first ORF codes for the γ-subunit (SEQ ID NO: 12).Further, the nucleotide sequence of a fragment containing thefull-length β-subunit gene is shown in SEQ ID NO: 15. Further, the aminoacid sequence of the β-subunit is shown in SEQ ID NO: 16. It is inferredthat the amino acid numbers 1 to 22 in SEQ ID NO: 16 correspond to asignal peptide. Although the first amino acid residues are Val in SEQ IDNOS: 15 and 16, they are very likely to be Met and may be eliminatedafter translation.

The mutant GDH of the present invention may consist of the α-subunitalone, a complex comprising the α-subunit and the β-subunit, or acomplex comprising the α-subunit, β-subunit and γ-subunit. The mutantGDH of the present invention is obtained by introducing a specificmutation into the α-subunit in any case, and may have a conservativemutation in addition to the above specific mutation. Further, the othersubunits may be of a wild type or have a conservative mutation. The term“conservative mutation” means a mutation that does not substantiallyaffect the GDH activity.

The mutant α-subunit of the present invention preferably has the aminoacid sequence of SEQ ID NO: 13 except that it includes the specificmutation described later. Further, the mutant α-subunit may have theaforementioned conservative mutation so long as it has the GDH activity.That is, it may be a protein having an amino acid sequence of SEQ ID NO:13 including substitution, deletion, insertion or addition of one ormore amino acid residues in addition to the aforementioned specificmutation. SEQ ID NO: 13 shows an amino acid sequence that can be encodedby the nucleotide sequence of SEQ ID NO: 11. However, the methionineresidue at the N-terminus may be eliminated after translation. Theaforementioned term “one or several” preferably means a number of 1 to10, more preferably 1 to 5, particularly preferably 1 to 3.

Further, the β-subunit typically has the amino acid sequence of SEQ IDNO: 16. However, so long as it functions as the β-subunit of GDH, it maybe a protein having an amino acid sequence of the amino acid numbers 23to 425 of SEQ ID NO: 16 including substitution, deletion, insertion oraddition of one or more amino acid residues. The aforementioned term“one or several” preferably means a number of 1 to 20, more preferably 1to 10, particularly preferably 1 to 5. The expression “functions as theGDH β-subunit” means to function as cytochrome C without degrading theenzymatic activity of GDH.

Specific examples of the wild type α-subunit gene include a DNAcontaining the nucleotide sequence corresponding to the nucleotidenumbers 764 to 2380 of SEQ ID NO: 11. Further, the α-subunit gene may bea DNA having the nucleotide sequence corresponding to the nucleotidenumbers 764 to 2380 in the nucleotide sequence of SEQ ID NO: 11 or a DNAwhich is hybridizable with a probe prepared from that sequence under astringent condition and codes for a protein having the GDH activity.

Further, specific examples of the β-subunit gene include a DNA havingthe nucleotide sequence corresponding to the nucleotide numbers 187 to1398 of SEQ ID NO: 9. Further, the β-subunit gene may be a DNA which hasthe nucleotide sequence corresponding to the nucleotide numbers 187 to1398 of SEQ ID NO: 9, or a DNA which is hybridizable with a probeprepared from that sequence under a stringent condition and codes for aprotein that can function as the β-subunit.

Examples of the aforementioned stringent condition include, for example,a condition under which DNAs having a homology of 70% or more,preferably 80% or more, more preferably 90% or more, particularlypreferably 95% or more, hybridize with each other, and it isspecifically exemplified by the condition of 1×SSC, 0.1% SDS at 60° C.

The α-subunit gene and the β-subunit gene can be obtained by, forexample, PCR using chromosomal DNA of the Burkhorderia cepacia KS1strain as a template. Primers for PCR can be prepared by chemicalsynthesis on the basis of the aforementioned nucleotide sequences.Further, they can also be obtained from chromosomal DNA of theBurkhorderia cepacia KS1 strain by hybridization using anoligonucleotide prepared on the basis of the aforementioned sequences asa probe. Further, variants thereof can also be similarly obtained fromother strains of Burkhorderia cepacia. Examples of the other bacterialstrains include the aforementioned JCM2800 and JCM2801 strains. Theα-subunits of GDHs produced by these strains have homologies of 95.4 and93.7%, respectively, to the α-subunit of the KS1 strain.

Further, even GDHs produced by other microorganisms can be used for theproduction of mutant GDH of the present invention so long as they have astructure and enzymological characteristics similar to those of GDHproduced by Burkhorderia cepacia.

In the mutant GDH of the present invention, substrate specificity toglucose is improved by introducing a specific mutation into theaforementioned wild type GDH. The expression “substrate specificity toglucose is improved” means that reactivity to other sugars such asmonosaccharides, disaccharide and oligosaccharides, for example,maltose, galactose, xylose and so forth, is decreased while thereactivity to glucose is substantially maintained, or reactivity toglucose is improved compared with reactivities to other sugars. Forexample, even if reactivity to glucose is decreased, but if reactivitiesto other sugars are decreased to a greater extent, substrate specificityto glucose is improved. Further, even if reactivities to other sugarsare increased, but if substrate specificity to glucose is increased to agreater extent, substrate specificity to glucose is improved.Specifically, for example, if the ratio of specific activity for glucoseand specific activity for another sugar, for example, maltose((reactivity to another sugar/reactivity to glucose)×100) is decreasedby 10% or more, preferably 20% or more, more preferably 50% or more,substrate specificity to L glucose is improved.

The aforementioned specific mutation means any one of amino acidsubstitution at position 472, amino acid substitution at position 475and amino acid substitution at both positions 472 and 475 in the aminoacid sequence of the SEQ ID NO: 13. More specific examples of themutation include amino acid substitutions described below. The numeralsshown below represent a position in the amino acid sequence, the aminoacid residues represent an amino acid residue after substitution at theaforementioned position, and “+” means that two amino acid substitutionsare simultaneously included. Among the following amino acidsubstitutions, amino acid substitutions at position 472 are listed in(A), amino acid substitutions at position 475 are listed in (B), andamino acid substitutions at both positions 472 and 475 are listed in(C). For example, “472Asn+475(Asp, Gly, His, Phe, Ser, Tyr)” meansmutations for substitution of Asn for the amino acid residue at position472 (Ala in the wild type), and substitution of Asp, Gly, His, Phe, Seror Tyr for the amino acid residue at position 475 (Asn in the wildtype).

-   (A) 472Arg, 472Asn, 472Asp, 472Cys, 472Glu, 472Gly, 472His, 472Ile,    472Leu, 472Met, 472Phe, 472Pro, 472Ser, 472Trp, 472Tyr, 472Val,-   (B) 475Asp, 475Cys, 475Glu, 475Gly, 475His, 475Met, 475Phe, 475Ser,    475Tyr, 475Val,-   (C) 472Arg+475(Asp, Glu, Gly, His, Phe, Ser, Tyr), 472Asn+475(Asp,    Gly, His, Phe, Ser, Tyr), 472Asp+475(H is, Phe, Ser, Val),    472Cys+475(Asp, Gly, His, Phe, Ser), 472Glu+475(Asp, Glu, Gly, His,    Phe, Ser, Tyr), 472Gly+475(Asp, Cys, Gly, Met, Phe, Ser, Tyr),    472His+475(Cys, Glu, His, Met, Phe, Ser, Tyr), 472Ile+475(Asp, Cys,    Glu, Gly, His, Met, Phe, Ser, Tyr), 472Leu+475(Asp, Gly, His, Phe,    Ser, Tyr), 472Met+475(Asp, Gly, His, Phe, Ser), 472Phe+475(Asp, Glu,    Gly, His, Met, Phe, Ser, Tyr), 472Pro+475His 472Ser+475(Asp, Glu,    Gly, His, Phe, Ser), 472Trp+475(H is, Phe, Ser), 472Tyr+475(Asp,    His, Phe, Ser), 472Val+475(Asp, Glu, Gly, His, Phe, Ser).

Among the aforementioned amino acid substitutions, preferred are listedbelow.

-   (D) 472Arg, 472Asn, 472Asp, 472Glu, 472Gly, 472Phe, 472Pro,-   (E) 475Asp, 475Cys, 475Glu, 475Gly, 475Met, 475Phe-   (F) 472Arg+475(Asp, Gly, His, Phe), 472Asn+475(Gly, His, Phe, Tyr),    472Asp+475(H is, Ser), 472Cys+475(Gly, His, Phe), 472Glu+475(Glu,    His, Phe, Tyr), 472Gly+475(Asp, Phe, Tyr), 472His+475(H is, Ser),    472Ile+475(Asp, Glu, Gly, His, Ser), 472Leu+475(Gly, His, Phe, Tyr),    472Met+475(Asp, Gly, His, Phe), 472Phe+475(Asp, Glu, Gly, His, Phe,    Ser, Tyr), 472Ser+475(Glu, Gly, His, Phe), 472Trp+475(H is, Phe),    472Tyr+475His, 472Val+475(Asp, Glu, Gly, His, Phe).

The positions of the aforementioned amino acid substitution mutationsare those in SEQ ID NO: 13, that is, the amino acid sequence of the wildtype GDH α-subunit of the Burkholderia cepacia KS1 strain, and in a GDHα-subunit homologue or variant having an amino acid sequence containingsubstitution, deletion, insertion or addition of one or more amino acidresidues in the amino acid sequence of SEQ ID NO: 13 in addition to theaforementioned specific mutations, the positions are those correspondingto the positions of aforementioned amino acid substitutions determinedby alignment with the amino acid sequence of SEQ ID NO: 13. For example,in a conservative GDH α-subunit variant having deletion of one aminoacid residue in the region of 1st to 471st positions, the 472nd and475th positions represent the 471st and 474th positions in the variant.

The inventors of the present invention investigated the region ofglucose dehydrogenase involved in binding to FAD and neighboring regionsas positions for introduction of the mutation for improving thesubstrate specificity. As the region involved in binding to FAD, the FADneighboring region (FAD-covering lid) or FAD-binding domain,specifically, regions corresponding to the amino acid sequences of SEQID NOS: 1 to 4, were contemplated.

The term “regions corresponding to amino acid sequences” means, in theGDH α-subunit of the Burkhorderia cepacia KS1 strain having the aminoacid sequence of SEQ ID NO: 13, regions having the amino acid sequenceof SEQ ID NO: 1, 2 or 4, that is, regions of the amino acid numbers 88to 92, 57 to 61, and 470 to 504 in SEQ ID NO: 13. Further, in the GDHα-subunit having an amino acid sequence homologous to the amino acidsequence of SEQ ID NO: 13, the regions are those corresponding to theregions of the amino acid numbers 88 to 92, 57 to 61 or 470 to 504 inthe GDH α-subunit of the aforementioned Burkhorderia cepacia KS1 straindetermined by alignment with the amino acid sequence of SEQ ID NO: 13.

The inventors of the present invention compared the amino acid sequencesof the GMC oxidoreductase family enzymes using FAD as a coenzyme,sorbitol dehydrogenase of Gluconobacter oxydans (GenBank accessionAB039821), 2-ketoglutarate dehydrogenase of Erwinia herbicola (GenBankaccession AF068066), cellobiose dehydrogenase (CDH) of Phanerochaetechrysosporium (J. Mol. Biol., 315(3), 421-34 (2002)), cholesteroloxidase (COD) of Streptomyces species (J. Struct. Biol. 116(2), 317-9(1996)), and glucose oxidase of Penicillium amagasakiens (Eur. J.Biochem. 252, 90-99 (1998)), and found a region in which the FAD-bindingdomain and FAD-covering lid were conserved and a region in which prolinewas conserved, which is an amino acid residue involved in folding ofproteins. Then, they examined the possibility of improving substratespecificity by modifying sequences in the vicinity of the bordersbetween these regions and other regions. As a result, they confirmedthat the substrate specificity could be improved by mutations of theaforementioned amino acid residues.

A GDH α-subunit having a desired mutation can be obtained by introducinga nucleotide mutation corresponding to a desired amino acid mutationinto a DNA coding for the GDH α-subunit (α-subunit gene) bysite-directed mutagenesis and expressing the obtained mutant DNA byusing a suitable expression system. Further, a mutant GDH complex can beobtained by expressing a DNA coding for the mutant GDH α-subunittogether with a DNA coding for the β-subunit (β-subunit gene) or theβ-subunit gene and a DNA coding for the γ-subunit (γ-subunit gene). Forthe introduction of a mutation into a DNA coding for the GDH α-subunit,a polycistronic DNA fragment coding for the GDH α-subunit, γ-subunit andβ-subunit in this order may also be used.

Substrate specificities to sugars of the GDH α-subunit or the GDHcomplex introduced with the mutation can be determined by examiningreactivities to various sugars by the methods described in the examplesand comparing them with reactivities of a wild type GDH α-subunit or awild type GDH complex.

A polycistronic DNA fragment coding for the γ-subunit, α-subunit andβ-subunit in this order can be obtained by, for example, PCR usingchromosomal DNA of the Burkhorderia cepacia KS1 strain as a template andoligonucleotides having the nucleotide sequences of SEQ ID NOS: 12 and13 as primers (see the examples described later).

Examples of vectors used for obtaining the genes of GDH subunits,introduction of mutation, expression of the genes and so forth includevectors that function in Escherichia bacteria, and specific examplesthereof include pTrc99A, pBR322, pUC18, pUC118, pUC19, pUC119, pACYC184,pBBR122 and so forth. Examples of the promoters used for expression ofgenes include lac, trp, tac, trc, P_(L), tet, PhoA and so forth.Further, insertion of these genes into a vector and ligation of apromoter can be performed in one step by inserting the α-subunit gene orother subunit genes at a suitable site in an expression vectorcontaining the promoter. Examples of such an expression vector includepTrc99A, pBluescript, pKK223-3 and so forth.

Further, the α-subunit gene or other subunit genes may be incorporatedinto chromosomal DNA of a host microorganism in an expressible form.

Examples of the method for transforming a microorganism with arecombinant vector include, for example, the competent cell method usinga calcium treatment, protoplast method, electroporation and so forth.

Examples of the host microorganism include Bacillus bacteria such asBacillus subtilits, yeast such as Saccharomyces cerevisiae andfilamentous fungi such as Aspergillus niger. However, the hostmicroorganism is not limited to these examples, and host microorganismssuitable for producing foreign proteins can be used.

The mutant α-subunit, the mutant GDH complex, and the microorganismexpressing them of the present invention can be used as an enzymeelectrode of a glucose sensor or a component of a glucose assay kit. Aglucose sensor and glucose assay kit using the wild type GDH ofBurkhorderia cepacia are described in U.S. Patent No. 2004/0023330A1.The mutant GDH of the present invention can also be used in a similarmanner.

EXAMPLES

The present invention will be explained more specifically with referenceto the following examples. However, the scope of the present inventionis not limited to these examples.

Example 1 Plasmids expressing GDH of Burkhorderia cepacia

As plasmids expressing GDH of Burkhorderia cepacia, a plasmid expressingthe GDH α-subunit and γ-subunit and a plasmid expressing the α-subunit,β-subunit and γ-subunit were prepared.

<1>Plasmid Expressing GDH α-Subunit and γ-Subunit

As a plasmid expressing the α-subunit and γ-subunit, plasmid pTrc99A/γ+αdescribed in WO02/036779 was used. This plasmid is a plasmid obtained byinserting a DNA fragment sequentially containing the GDH γ-subunitstructural gene and the α-subunit structural gene isolated fromchromosomal DNA of the Burkhorderia cepacia KS1 strain (FERM BP-7306)into the vector pTrc99A (Pharmacia) at the NcoI/HindIII site as acloning site thereof. The GDHγα gene in this plasmid is regulated by thetrc promoter. pTrc99A/γ+α has an ampicillin resistance gene.

<2>Plasmid Expressing GDH α-Subunit, β-Subunit and γ-Subunit

A plasmid expressing the GDH α-subunit, β-subunit and γ-subunit wasprepared as follows.

(1) Preparation of Chromosomal DNA from Burkhorderia cepacia KS1 Strain

A chromosomal gene was prepared from the Burkhorderia cepacia KS1 strainin a conventional manner. That is, the TL liquid medium (10 g ofpolypeptone, 1 g of yeast extract, 5 g of NaCl, 2 g of KH₂PO₄, 5 g ofglucose in 1 L, pH 7.2) was used, and cells of the strain was shakenovernight in the medium at 34° C. The grown cells were collected bycentrifugation. The cells were suspended in a solution containing 10 mMNaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% SDS and 100 μg/ml ofproteinase K and treated at 50° C. for 6 hours. To the mixture was addedan equal volume of phenol-chloroform, and the mixture was stirred atroom temperature for 10 minutes. Then, the supernatant was collected bycentrifugation. To the supernatant was added sodium acetate at a finalconcentration of 0.3 M, and 2-fold volume of ethanol was overlaid toprecipitate chromosomal DNA in the intermediate layer. The DNA wascollected with a glass rod, washed with 70% ethanol, and then dissolvedin a suitable volume of TE buffer to obtain a chromosomal DNA solution.

(2) Preparation of DNA Fragment Coding for GDH γ-subunit, α-subunit andβ-subunit

A DNA fragment coding for the GDH γ-subunit, α-subunit and β-subunit wasamplified by PCR using the aforementioned chromosomal DNA as a templateand oligonucleotides having the following sequences as primers.

[Forward primer] (SEQ ID NO: 5) 5′-CATGCCATGGCACACAACGACAACAC-3′[Reverse primer] (SEQ ID NO: 6) 5′-GTCGACGATCTTCTTCCAGCCGAACATCAC-3′

The C-terminus side of the amplified fragment was blunt-ended, theN-terminus side was digested with NcoI, and the fragment was ligated tosimilarly treated pTrc99A (Pharmacia). E. coli DH5α was transformed withthe obtained recombinant vector, and colonies grown on the LB agarmedium containing 50 μg/mL of ampicillin were collected. The obtainedtransformants were cultured in the liquid LB medium, plasmids wereextracted, and DNA fragments inserted in the plasmids were analyzed. Asa result, an inserted fragment of about 3.8 kb was confirmed. Thisplasmid was designated as pTrc99Aγαβ. The structural genes of GDH inthis plasmid are regulated by the trc promoter. pTrc99Aγαβ has anampicillin resistance gene and a kanamycin resistance gene.

Example 2 Introduction of Mutation into GDH α-subunit Gene

By using a commercially available site-directed mutatgenesis kit(QuikChangeII Site-Directed Mutagenesis Kit, Stratagene), the codon ofaspartic acid (GAT) or glutamic acid (GAA) was substituted for the codonof the 475th asparagine (AAT) in the GDH α-subunit gene contained in theplasmids pTrc99A/γ+α and pTrc99Aγαβ described in Example 1. As primers,the following oligonucleotides were used. Hereinafter, substitution ofan aspartic acid residue for the 475th asparagine residue is referred toas “Asn475Asp”, and substitution of a glutamic acid residue for the475th asparagine residue is referred to as “Asn475Glu”.

Primers for Asn475Asp substitution [Forward primer] (SEQ ID NO: 7)5′-CGCGCCGAACGATCACATCACGGGC-3′ [Reverse primer] (SEQ ID NO: 8)5′-GCCCGTGATGTGATCGTTCGGCGCG-3′ Primers for Asn475Glu substitution[Forward primer] (SEQ ID NO: 9) 5′-GAATTCGCGCCGAACGAACACATCAGGGGCTCG-3′[Reverse primer] (SEQ ID NO: 10) 5′-CGAGCCCGTGATGTGTTCGTTCGGCGCGAATTC-3′

PCR was performed by using the following reaction composition. After areaction at 95° C. for 30 seconds, a cycle of reactions at 95° C. for 30seconds, 55° C. for 1 minute and 68° C. for 8 minutes was repeated 15times. Then, after a reaction at 68° C. for 30 minutes, the reactionmixture was maintained at 4° C.

[Reaction mixture composition] Template DNA (5 ng/μl) 2 μl (pTrc99A/γ +α and pTrc99Aγαβ) 10× Reaction buffer 5 μl Forward primer (100 ng/μl)1.25 μl Reverse primer (100 ng/μl) 1.25 μl dNTP 1 μl Distilled water38.5 μl DNA polymerase 1 μl Total 50 μl

After PCR, 0.5 μl of DNA polymerase I was added to the reaction mixture,and the mixture was incubated at 37° C. for 1 hour to decompose thetemplate plasmid.

Competent cells of Escherichia coli DH5α (supE44, ΔlacU169(φ+80lacZΔM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1) weretransformed with the obtained reaction mixture. Plasmid DNA was preparedfrom several colonies grown on the LB agar medium (1% bacto tryptone,0.5% yeast extract, 1% sodium chloride, 1.5% agar) containing ampicillin(50 μg/ml) and kanamycin (30 μg/ml), and sequence analysis was performedto confirm that the objective mutations had been introduced into the GDHα-subunit gene. pTrc99A/γ+α and pTrc99Aγαβ introduced with the Asn475Aspmutation were designated as pTrcγαAsn475Asp and pTrcγαβAsn475Asp,respectively. Further, pTrc99A/γ+α and pTrc99Aγαβ introduced with theAsn475Glu mutation were designated as pTrcγαAsn475Glu andpTrcγαβAsn475Glu, respectively.

Example 3 Analysis of Substrate Specificity of Mutant GDHs

Mutant GDHs were produced by using the mutant GDH expressing plasmidsobtained in Example 2, and substrate specificities thereof wereexamined.

(1) Culture

The Escherichia coli DH5a strain introduced with pTrcγαAsn475Glu andpTrcγαβAsn475Glu were each cultured overnight at 37° C. in 2 ml of theLB medium (containing 50 μg/ml of ampicillin and 30 μg/ml of kanamycin)in an L-shaped tube with shaking. These culture broths were inoculatedin 150 ml of the LB medium (containing 50 μg/ml of ampicillin and 30μg/ml of kanamycin) contained in a 500-ml Sakaguchi flask, and the cellswere cultured at 37° C. with shaking. After 3 hours from the start ofculture, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a finalconcentration of 0.1 mM, and the cells were further cultured for 2hours.

(2) Preparation of Enzyme Samples

The cells were collected from each culture broth obtained as describedabove, washed, then suspended in 10 mM potassium phosphate buffer (PPB,pH 7.0) containing 1 ml of 0.2% Triton X-100 per 0.3 mg of wet cells,and disrupted by ultrasonication. This suspension was centrifuged (10000rpm, 10 min, 4° C.) to remove the residues, then the supernatant wasultracentrifuged (50,000 r.p.m., 60 min, 4° C.), and the obtainedsupernatant (water-soluble fraction) was used as a crude enzyme sample.Further, this sample was purified by usual hydrophobic chromatography(column: Octyl Sepharose, Amersham Biosciences) and ion exchangechromatography (Q-Sepharose, Amersham Biosciences) to obtain a purifiedenzyme sample. The objective enzyme fraction was determined by using GDHactivity as an index.

(3) Measurement of GDH Activity

To 8 μl of the aforementioned purified enzyme sample was added 8 μl of areagent for measuring activity (solution obtained by adding 10 mM PPBcontaining 0.2% (w/v) Triton X-100 to 12 μl of 600 mM methylphenazinemethosulfate (PMS) and 120 μl of 6 mM 2,6-dichrolophenol-indophenol(DCIP) to make a total volume of 480 μl). This mixture was preincubatedat each reaction temperature for one minute by using an aluminum blockthermostatic chamber, then 8 μl of a substrate (glucose or maltose) ateach concentration or distilled water was quickly added to the mixture,and the mixture was stirred. Absorbance at 600 nm as the DCIP-originatedabsorption wavelength was measured by using a spectrophotometer. Thefinal concentrations of the reagents, DCIP and PMS, were 0.06 and 0.6mM, respectively. The final concentrations of the substrate were 40, 20,10 and 5 mM.

The results are shown in Table 1 and FIGS. 1 to 5.

TABLE 1 Substrate concentration Activity (U/ml) mM Glucose MaltosepTrc99A/γ + α 40 1.65 1.01 20 1.70 1.03 10 1.71 1.07 5 1.57 0.72pTrcγαAsn475Asp 40 19.46 1.30 20 7.75 0.67 10 4.15 0.21 5 2.53 0.00pTrc99Aγαβ 40 5.49 1.82 20 5.38 1.78 10 5.03 1.57 5 4.20 1.24pTrcγαβAsn475Asp 40 11.57 1.35 20 6.53 0.93 10 3.21 0.39 5 2.50 0.13pTrcγαβAsn475Glu 40 8.62 2.10 20 7.13 1.26 10 6.12 0.84 5 4.95 0.43

As clearly seen form these results, it is evident that all the mutantGDHs have reduced reactivity to maltose while maintaining reactivity toglucose, that is, their specificity to glucose is improved.

Example 4 Introduction of Mutation into GDH α-Subunit Gene

Mutations were introduced into the GDH α-subunit gene contained inpTrc99Aγαβ obtained in Example 1 at the 475th position and neighboringpositions, and substrate specificity of the mutant enzymes wasevaluated. Mutations were introduced in the same manner as in Example 2.Primers for introducing mutations were prepared as follows. In the basicprimers (wild type) shown in FIG. 6 (forward primer: SEQ ID NO: 17,reverse primer: SEQ ID NO: 18), codons were changed at predeterminedpositions (472nd and 475th) as shown in the codon change table mentionedin FIG. 6 to prepare primers for introducing various mutations.

Example 5 Analysis of Substrate Specificity of Mutant GDHs

Mutant GDHs were produced in the same manner as in Example 3 by usingthe mutant GDH expressing plasmids obtained in Example 4, and substratespecificities thereof were examined. The enzymatic activity was examinedby using crude enzyme samples. The specific activity for glucose,specific activity for maltose and reaction ratio (specific activity formaltose/specific activity for glucose, unit is U/ml.) of each mutant GDHare shown in Tables 2 to 7. When the specific activity for glucose was0.5 U/ml or lower, it was judged as no activity, and such a result wasindicated with “−” in the tables.

As a result, for the 475th position, it was confirmed that substitutionsother than the substitution of aspartic acid (GAT) or glutamic acid(GAA) for asparagine performed in Example 2 also had an effect ofimproving the substrate characteristics. Further, it was found thatsubstitution of another amino acid for asparagine (AAC) at the 472ndposition in the vicinity of the 475th position could also improve thesubstrate characteristics. Further, it was also found that a combinationof the amino acid substitutions at the 472nd and 475th positions couldsynergistically improve the substrate characteristics.

TABLE 2 substrate conc.: 10 mM specific activity to glucose (U/ml broth)475 → 472 ↓ Ala Arg Asn Asp Cys Glu Gln Gly His Ile Ala — — 7 6.5 24 7 —4.75 0.85 — Arg — — 6 2.35 — 1.5 — 2.3 2.4 — Asn — — 6.2 0.75 — — — 2.34.7 — Asp — — 1.35 — — — — — 1.75 — Cys — — 6.45 0.75 — — — 4 4.3 — Glu— — 6.1 2.15 — 1.2 — 3.45 4.65 — Gln — — — — — — — — — — Gly — — 6.350.6 1 — — 6.85 — — His — — 4.45 — 0.85 2.75 — — 4.4 — Ile — — 7.25 2.40.75 2.2 — 2.1 4.4 — Leu — — 6.35 0.75 — — — 1.9 5.65 — Lys — — — — — —— — — — Met — — 5.9 1.85 — — — 3.3 5.8 — Phe — — 6.79 0.65 — 0.55 — 1.756.25 — Pro — — 1.2 — — — — — 2.3 — Ser — — 6.1 1.1 — 2.45 — 4.25 4 — Thr— — — — — — — — — — Trp — — 4.55 — — — — — 6.3 — Tyr — — 4.35 0.5 — — —— 5.75 — Val — — 5.75 2.2 — 0.85 — 2.5 5.9 —

TABLE 3 substrate conc.: 10 mM specific activity to maltose (U/ml broth)475 → 472 ↓ Ala Arg Asn Asp Cys Glu Gln Gly His Ile Ala — — 3.5 1 0.551.5 — 1.35 0.8 — Arg — — 0.85 0.25 — 0.35 — 0.25 0.35 — Asn — — 0.750.21 — — — 0.28 0.44 — Asp — — 0.1 — — — — — 0.25 — Cys — — 1.2 0.18 — —— 0.2 0.41 — Glu — — 0.7 0.85 — 0.15 — 0.35 0.41 — Gln — — — — — — — — —— Gly — — 0.8 0.08 0.3 — — 1.55 — — His — — 1.05 — 0.2 0.5 — — 0.5 — Ile— — 0.85 0.2 0.1 0.2 — 0.33 0.45 — Leu — — 1.2 0.2 — — — 0.3 0.45 — Lys— — — — — — — — — — Met — — 1.1 0.25 — — — 0.34 0.45 — Phe — — 0.81 0.09— 0.09 — 0.25 0.45 — Pro — — 0.15 — — — — — 0.6 — Ser — — 1.25 0.24 —0.25 — 0.5 0.4 — Thr — — — — — — — — — — Trp — — 1.1 — — — — — 0.6 — Tyr— — 1.05 0.14 — — — — 0.35 — Val — — 1.1 0.2 — 0.1 — 0.22 0.49 —

TABLE 4 substrate conc.: 10 mM maltose/glucose (reaction ratio) 475 →472 ↓ Ala Arg Asn Asp Cys Glu Gln Gly His Ile Ala — — 50% 15% 23% 21% —28% 94% — Arg — — 14% 10% — 23% — 11% 14% — Asn — — 12% 27% — — — 12% 9% — Asp — —  7% — — — — — 14% — Cys — — 19% 24% — — —  5% 10% — Glu —— 11% 40% — 13% — 10%  9% — Gln — — — — — — — — — — Gly — — 13% 13% 30%— — 23% — — His — — 24% — 24% 18% — — 11% — Ile — — 12%  8% 13%  9% —16% 10% — Leu — — 19% 27% — — — 16%  8% — Lys — — — — — — — — — — Met —— 19% 14% — — — 10%  8% — Phe — — 12% 13% — 15% — 14%  7% — Pro — — 13%— — — — — 26% — Ser — — 20% 22% — 10% — 12% 10% — Thr — — — — — — — — —— Trp — — 24% — — — — — 10% — Tyr — — 24% 27% — — — —  6% — Val — — 19% 9% — 12% —  9%  8% — Total: 60

TABLE 5 substrate conc.: 10 mM specific activity to glucose (U/ml broth)475 → 472 ↓ Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Ala — — 1.6 1.2 —0.3 — — 1.05 1.15 Arg — — — 4.1 — 4.25 — — 2 — Asn — — — 2.5 — 5.25 — —1.3 — Asp — — — 2.65 — 3 — — — 6.2 Cys — — — 2.85 — 4.5 — — — — Glu — —— 1.4 — 6 — — 1.9 — Gln — — — — — — — — — Gly — — 3.1 3.25 — 8 — — 0.5 —His — — 1.1 1.9 — 6.6 — — — — Ile — — 2.7 2.95 — 7 — — 3 — Leu — — — 1.6— 5.5 — — 2 — Lys — — — — — — — — — Met — — — 3.25 — 4.6 — — — — Phe — —1.7 0.75 — 10.5 — — 1.55 — Pro — — — — — — — — — Ser — — — 2.5 — 5.5 — —— — Thr — — — — — — — — — Trp — — — 3.5 — 2.15 — — — — Tyr — — — 0.85 —3.85 — — — — Val — — — 3.5 — 7 — — 2.8 —

TABLE 6 substrate conc.: 10 mM specific activity to maltose (U/ml broth)475 → 472 ↓ Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Ala — — 0.5 0.35 —0.25 — — 0.65 1.05 Arg — — 0.26 — 1.7 — — 0.33 Asn — — 0.45 — 1.1 — —0.18 Asp — — 0.75 — 0.4 — — 3.3 Cys — — 0.2 — 0.95 — — Glu — — 0.15 —1.15 — — 0.07 Gln — — — — — Gly — — 0.55 0.4 — 5 — — 0.05 His — — 0.220.35 — 0.75 — — Ile — — 0.95 0.6 — 0.5 — — 0.7 Leu — — 0.12 — 1.3 — —0.25 Lys — — — — — Met — — 0.2 — 1.1 — — Phe — — 0.75 0.07 — 0.65 — —0.2 Pro — — — — — Ser — — 0.2 — 1.3 — — Thr — — — — — Trp — — 0.2 — 0.55— — Tyr — — 0.15 — 1.1 — — Val — — 0.2 — 1.2 — —

TABLE 7 substrate conc.: 10 mM maltose/glucose (reaction ratio) 475 →472 ↓ Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Ala — — 31% 29% — 83% — —62% 91% Arg — — —  6% — 40% — — 16% — Asn — — — 18% — 21% — — 13% — Asp— — — 28% — 13% — — — 53% Cys — — —  7% — 21% — — — — Glu — — — 11% —19% — —  3% — Gln — — — — — — — — — — Gly — — 18% 12% — 63% — — 10% —His — — 20% 18% — 11% — — — — Ile — — 35% 20% —  7% — — 23% — Leu — — — 8% — 24% — — 13% — Lys — — — — — — — — — — Met — — —  6% — 24% — — — —Phe — — 44%  9% —  6% — — 13% — Pro — — — — — — — — — — Ser — — —  8% —24% — — — — Thr — — — — — — — — — — Trp — — —  6% — 26% — — — — Tyr — —— 18% — 29% — — — — Val — — —  6% — 17% — — — — Total: 60

Example 6 Evaluation of Purified Enzymes Based on SV Plot

SV plots were obtained for several mutant GDHs which showed improvedsubstrate specificity in Example 5. Each mutant GDH was purified in thesame manner as in Example 3. The results are shown in FIGS. 7 and 8 andTable 8.

As a result, it was confirmed that the reaction ratios (specificactivity for maltose/specific activity for glucose) of the purifiedenzymes were also improved and became lower than that of the wild typeat all the examined substrate concentrations. Further, since the resultswere substantially consistent with the measurement results using thecrude enzyme solutions in Example 5, sufficient feasibility of screeningfor modified enzymes using crude enzymes could be confirmed. Further,modified enzymes to be used for a glucose sensor were selected fromthese candidates. For this purpose, since the blood maltose levelelevates up to 200 mg/dl even at most, attentions were paid particularlyto the reaction ratios at the substrate concentrations of 180 and 90mg/dl. As a result, 472Asp475His was selected as a candidate of whichreactivity to glucose was not so decreased compared with the wild type,and 472Glu475Tyr was selected as a candidate of which reactivity toglucose decreased but hardly reacted with maltose.

TABLE 8 Evaluation of characteristics of enzymes U/mg-p substrate conc.1440 720 360 180 90 45 22.5 11.25 mg/dl reactivity to glucose wild-type2198.8 2175.0 2035.8 1750.7 1264.6 803.7 461.7 244.0 472F 1123.3 1004.7824.7 484.0 280.3 128.4 40.2 5.4 472D475F 811.5 678.6 512.5 328.9 215.3107.3 33.7 4.1 475D 1324.4 1025.2 730.2 460.7 275.5 136.1 59.1 16.7472D475H 2979.1 2522.1 1978.3 1322.9 795.0 430.8 207.1 82.9 472E475F2153.8 1600.9 1079.9 667.3 366.0 165.6 46.6 7.0 472E475Y 734.4 466.8219.5 88.2 17.4 2.4 0.8 0.2 475E 1296.4 768.2 426.0 209.6 reactivity tomaltose wild-type 975.5 763.8 532.1 323.9 157.3 59.7 14.1 1.3 472F 215.4133.6 58.9 10.3 2.3 0.6 0.3 0.3 472D475F 265.4 138.9 51.2 7.7 1.2 0.30.2 0.2 475D 290.1 197.4 116.2 48.8 13.0 1.4 0.4 0.2 472D475H 342.6228.5 131.9 59.6 17.1 3.6 0.9 0.4 472E475F 544.1 304.8 137.2 39.7 5.51.4 0.4 0.2 472E475Y 23.3 4.2 1.3 0.4 0.3 0.1 0.2 0.0 475E 193.9 73.014.3 1.5 reaction ratio: maltose/glucose wild-type 44.4% 35.1% 26.1%18.5% 12.4% 7.4% 3.1% 0.5% 472F 19.2% 13.3% 7.1% 2.1% 0.8% 0.5% 0.6%4.7% 472D475F 32.7% 20.5% 10.0% 2.3% 0.6% 0.3% 0.5% 3.8% 475D 21.9%19.3% 15.9% 10.6% 4.7% 1.1% 0.6% 1.0% 472D475H 11.5% 9.1% 6.7% 4.5% 2.1%0.8% 0.4% 0.4% 472E475F 25.3% 19.0% 12.7% 5.9% 1.5% 0.8% 1.0% 2.5%472E475Y 3.2% 0.9% 0.6% 0.5% 1.7% 3.5% 25.0% 0.0% 475E 15.0% 9.5% 3.4%0.7%

Example 7 Preparation of Calorimetric Sensor for Measuring Blood SugarLevels using Mutant GDHs

Colorimetric sensors for measuring blood sugar level were prepared byusing 472Asp+475His type mutant GDH and 472Glu+475Tyr type mutant GDH.

A glucose sensor (1) having a basic structure shown in FIG. 9 wasprepared. That is, the aforementioned glucose sensor had a configurationthat a transparent cover (4) (material: PET) was laminated on atransparent base plate (2) via a spacer (3), and the capillary (5) wasdefined by the elements (2) to (4). The dimension of the capillary (5)was 1.3 mm×9 mm×50 μm (FIG. 9). The transparent base plate (2) and thetransparent cover (4) were formed with PET having a thickness of 250 μm,and the spacer (3) was formed with a black double-sided tape.

The glucose sensor had a first reagent part (1), a second reagent part(2) and a third reagent part (3) shown in FIG. 10, and ingredients andcoating amounts for each part are shown in Table 9. In the table, “Ru”represents a ruthenium hexaammine complex (Ru(NH₃)₆Cl₃), CHAPSrepresents 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid,ACES represents N-(2-acetamido)-2-aminoethanesulfonic acid, and MTTrepresents 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide.

TABLE 9 First reagent part Material solution for reagent part containingelectron transfer substance (solvent is water) Ru coating 200 mM 0.2 ulSecond reagent part Material solution for reagent part containing enzyme(solvent is water) Enzyme sucrose ACES coating enzymes conc. CHAPSmonolaurate (pH 7.5) amount wild type 15 KU/ml 0.20% 0.05% 75 mM 0.1 ul472D475H 15 KU/ml 0.20% 0.05% 75 mM 0.1 ul 472E475Y 15 KU/ml 0.20% 0.05%75 mM 0.1 ul Third reagent part Material solution for reagent partcontaining color developer (solvent is water) MTT acrylamide methanolcoating amount 60 mM 0.40% 50% 0.2 ul

An assay sample was supplied to the capillary of the aforementionedglucose sensor, and thereafter absorbance was repeatedly measured every0.1 second to prepare a time course of absorbance. For each measurementof absorbance, the third reagent part (3) was irradiated with lightalong the direction of the height of the capillary, and light thattransmitted through the glucose sensor was received upon theirradiation. The light irradiation was attained by irradiation withlight of 630 nm using a light-emitting diode. The transmitted light wasreceived with a photodiode.

As assay sample, blood added with glucose was used. Blood samples ofwhich hematocrit was adjusted to 42% added with glucose atconcentrations of 0, 100, 200 and 400 mg/dl were used to evaluatelinearity of the glucose sensor. The results are shown in FIGS. 11 (wildtype), 12 (472Glu475Tyr) and 13 (472Asp475His).

Further, blood samples of which hematocrit was adjusted to 42% andglucose concentration was adjusted to 45 mg/dl was further added withmaltose at concentrations of 0, 100, 200 and 300 mg/dl, and used toevaluate influence of maltose. The results are shown in FIGS. 14 (wildtype), 15 (472Glu475Tyr) and 16 (472Asp475His).

When maltose was added to the samples of 45 mg/dl of glucose, absorbanceincreased in a maltose concentration-dependent manner for the wild type,which suggested strong reaction with maltose. On the other hand, withthe sensors using the mutant enzymes, the maltoseconcentration-dependent increase of the absorbance was suppressed,showing less influence of maltose. The results obtained by convertingthese data into apparent blood sugar elevation values are shown in FIG.17. In the sensor using the wild type enzyme, a hypoglycemic level (45mg/dl of glucose) is apparently shown as a normal value (122 mg/dl ofglucose) due to contamination of maltose. On the other hand, when thesensor using the modified GDHs is used, the apparent blood sugar leveldoes not elevate to the normal range even when the sample iscontaminated with up to 300 mg/dl of maltose.

As clearly seen from the above results, in the glucose sensors using themutant GDHs, reactivity to maltose was significantly decreased eventhough linearity was maintained to an extent comparable to that of thewild type. If these glucose sensors using the mutant GDHs are used, ahypoglycemic value (50 mg/dl or less) is not judged as a normal value orhyperglycemic level even at a maltose blood concentration of the upperlimit (200 mg/dl) for administration at hospital or the like or higher,and thus safe therapeutic treatment can be conducted. Further, sinceGDHs do not react with dissolved oxygen as described above, accuratediagnosis and treatment of diabetic patients can be conducted byproviding sensors using these mutant GDHs.

Example 8 Verification of Effect of Combination of Amino AcidSubstitution at 472nd Position and Amino Acid Substitution at Positionother than 475th Position

In a mutant GDH having 472Phe type substitution, substitution ofphenylalanine was further introduced at positions in the vicinity of the475th position (477th to 497th positions) and randomly selectedpositions far from the 475th position (53rd to 73rd positions).

Mutations were introduced in the same manner as in Example 2 by usingpTrc99Aγαβ expressing a mutant GDH containing substitution ofphenylalanine at the 472nd position. The sequences of the forwardprimers used for the introduction of mutations are shown in Tables 10and 11. The sequences of the reverse primers were completelycomplementary strands of the forward primers.

TABLE 10 Mutation SEQ ID NO: I477F 19 T478F 20 G479F 21 S480F 22 T481F23 I482F 24 M483F 25 G484F 26 A485F 27 D486F 28 A487F 29 R488F 30 D489F31 S490F 32 V491F 33 V492F 34 D493F 35 K494F 36 D495F 37 C496F 38 R497F39

TABLE 11 Mutation SEQ ID NO: R53F 40 N54F 41 Q55F 42 P56F 43 D57F 44K58F 45 M59F 46 D60F 47 M62F 48 A63F 49 P64F 50 Y65F 51 P66F 52 S67F 53S68F 54 P69F 55 W70F 56 A71F 57 P72F 58 H73F 59

The results are shown in Tables 12 and 13. As clearly seen from theseresults, with combinations of amino acid substitution of 472Phe andsubstitution at positions other than the 475th position, activity waslost, no change occurred, or only an effect of increasing the reactivityto maltose was observed, and thus it was confirmed that the improvingeffect was not necessarily obtained by introducing mutations at anyarbitrary positions.

TABLE 12 10 mM 10 mM Mal/Glu mutated substituting Glucose Maltosereaction site amino acid U/ml U/ml ratio 472F None 6.79 0.81   12% 472F+475 F(Phe) 0.75 0.07  8.7% 472F+ 477 F(Phe) 0.11 0.16 inactive 472F+ 478F(Phe) 0.12 0.12 inactive 472F+ 479 F(Phe) 0.21 0.21 inactive 472F+ 480F(Phe) 0.26 0.30 inactive 472F+ 481 F(Phe) 0.10 0.08 inactive 472F+ 482F(Phe) 0.06 0.08 inactive 472F+ 483 F(Phe) 4.32 0.68 15.6% 472F+ 484F(Phe) 0.10 0.10 inactive 472F+ 485 F(Phe) 0.18 0.24 inactive 472F+ 486F(Phe) 1.26 0.40 32.0% 472F+ 487 F(Phe) 0.22 0.23 inactive 472F+ 488F(Phe) 2.85 0.65 22.9% 472F+ 489 F(Phe) 1.81 0.56 31.2% 472F+ 490 F(Phe)0.18 0.19 inactive 472F+ 491 F(Phe) 0.23 0.23 inactive 472F+ 492 F(Phe)0.19 0.24 inactive 472F+ 493 F(Phe) 0.15 0.15 inactive 472F+ 494 F(Phe)1.15 0.29 25.0% 472F+ 495 F(Phe) 0.29 0.23 inactive 472F+ 496 F(Phe)0.16 0.18 inactive 472F+ 497 F(Phe) 0.14 0.16 inactive

TABLE 13 10 mM 10 mM Mal/Glu mutated substituting Glucose Maltosereaction site amino acid U/ml U/ml ratio 472F None 6.79 0.81 11.9% 472F+475 F(Phe) 0.75 0.07 8.7% 472F+ 53 F(Phe) 3.44 0.48 13.8% 472F+ 54F(Phe) 3.00 0.54 18.2% 472F+ 55 F(Phe) 3.81 0.72 18.8% 472F+ 56 F(Phe)4.89 0.57 11.7% 472F+ 57 F(Phe) 2.41 0.41 17.2% 472F+ 58 F(Phe) 3.330.45 13.5% 472F+ 59 F(Phe) 4.07 0.58 14.2% 472F+ 60 F(Phe) 1.55 0.3723.7% 472F+ 62 F(Phe) 1.31 0.26 19.9% 472F+ 63 F(Phe) 2.91 0.44 15.0%472F+ 64 F(Phe) 0.54 0.28 51.4% 472F+ 65 F(Phe) 5.52 0.76 13.8% 472F+ 66F(Phe) 1.63 0.35 21.8% 472F+ 67 F(Phe) 3.91 0.48 12.3% 472F+ 68 F(Phe)4.32 0.86 19.8% 472F+ 69 F(Phe) 4.79 0.82 17.1% 472F+ 70 F(Phe) 5.340.64 12.0% 472F+ 71 F(Phe) 1.26 0.28 22.5% 472F+ 72 F(Phe) 3.65 0.5013.8% 472F+ 73 F(Phe) 1.20 0.26 21.3%

INDUSTRIAL APPLICABILITY

The mutant GDH of the present invention has improved substratespecificity to glucose and can be suitably used for measurement ofglucose using a glucose sensor or the like.

1. An isolated mutant glucose dehydrogenase exhibiting improvedsubstrate specificity to glucose, which is (1) a mutant of the proteincomprising the amino acid sequence of SEQ ID NO: 13, wherein said mutantconsists of amino acid substitution(s) at positions 472 and/or 475 aslisted below in (A)-(C); or (2) a mutant of the protein comprising theamino acid sequence of SEQ ID NO: 13 wherein said mutant consists ofamino acid substitution(s) at positions 472 and/or 475 as listed belowin (A)-(C) and consists of substitution, deletion, insertion or additionof one to ten amino acid residues at position(s) other than thepositions listed below and wherein mutants (1) or (2) have glucosedehydrogenase activity (numerals represent a position in the amino acidsequence, the amino acid residues represent an amino acid residue aftersubstitution at the position, and “+” means that two amino acidsubstitutions are simultaneously included), wherein improved substratespecificity for mutants (1) or (2) is a decrease of at least 10% in theratio of reactivity to maltose/reactivity to glucose compared to theglucose dehydrogenase of SEQ ID NO: 13: (A) 472Arg, 472Asn, 472Asp,472Cys, 472Glu, 472Gly, 472His, 472lle, 472Leu, 472Met, 472Phe, 472Pro,472Ser, 472Trp, 472Tyr, or 472Val; (B) 475Asp, 475Cys, 475Glu, 475Gly,475His, 475Met, 475Phe, 475Ser, 475Tyr, or 475Val; or (C) 472Arg+475(Asp, Glu, Gly, His, Phe, Ser, Tyr), 472Asn +475(Asp, Gly, His, Phe,Ser, Tyr), 472Asp +475(His, Phe, Ser, Val), 472Cys +475(Asp, Gly, His,Phe, Ser), 472Glu +475(Asp, Glu, Gly, His, Phe, Ser, Tyr), 472Gly+475(Asp, Cys, Gly, Met, Phe, Ser, Tyr), 472His +475(Cys, Glu, His, Met,Phe, Ser, Tyr), 472lle +475(Asp, Cys, Glu, Gly, His, Met, Phe, Ser,Tyr), 472Leu +475(Asp, Gly, His, Phe, Ser, Tyr), 472Met +475(Asp, Gly,His, Phe, Ser), 472Phe +475(Asp, Glu, Gly, His, Met, Phe, Ser, Tyr),472Pro +475His 472Ser +475(Asp, Glu, Gly, His, Phe, Ser), 472Trp+475(His, Phe, Ser), 472Tyr +475(Asp, His, Phe, Ser), or 472Val+475(Asp, Glu, Gly, His, Phe, Ser).
 2. The isolated mutant glucosedehydrogenase according to claim 1, which consists of amino acidsubstitution(s) at positions 472 and/or 475 selected from (A) to (C). 3.The isolated mutant glucose dehydrogenase according to claim 1, whereinthe substitutions at positions 472 and/or 475 consist of the amino acidsubstitution(s) listed in (D) to (F): (D) 472Arg, 472Asn, 472Asp,472Glu, 472Gly, 472Phe, or 472Pro, (E) 475Asp, 475Cys, 475Glu, 475Gly,475Met, or 475Phe, or (F) 472Arg +475(Asp, Gly, His, Phe), 472Asn+475(Gly, His, Phe, Tyr), 472Asp +475(His, Ser), 472Cys +475(Gly, His,Phe), 472Glu +475(Glu, His, Phe, Tyr), 472Gly +475(Asp, Phe, Tyr),472His +475(His, Ser), 472lle +475(Asp, Glu, Gly, His, Ser), 472Leu+475(Gly, His, Phe, Tyr), 472Met +475(Asp, Gly, His, Phe), 472Phe+475(Asp, Glu, Gly, His, Phe, Ser, Tyr), 472Ser +475(Glu, Gly, His,Phe), 472Trp +475(His, Phe), 472Tyr +475His, or 472Val +475(Asp, Glu,Gly, His, Phe).
 4. An isolated glucose dehydrogenase, which is (1) amutant of the protein comprising the amino acid sequence of SEQ ID NO:13, wherein said mutant consists of amino acid substitution(s) atpositions 472 and/or 475, or (2) a mutant of the protein comprising theamino acid sequence of SEQ ID NO: 13, wherein said mutant consists ofamino acid substitution(s) at positions 472 and/or 475 and consists ofsubstitution, deletion, insertion or addition of one to ten amino acidresidues at position other than positions 472 and/or 475, whereinmutants 1 or 2 have a glucose dehydrogenase activity, and wherein: (i)the substitutions at positions 472 and/or 475 in mutants (1) or (2)consist of replacement of at least either the arginine residue atposition 472 or the asparagine residue at position 475 in the amino acidsequence of SEQ ID NO: 13 with another amino acid residue, and (ii) aratio of specific activity for glucose and specific activity for maltose((reactivity to maltose/reactivity to glucose)×100) of the glucosedehydrogenase of mutants (1) or (2) is reduced by 10% or more comparedwith that of the glucose dehydrogenase of SEQ ID NO:
 13. 5. A mutantglucose dehydrogenase complex comprising at least the isolated mutantglucose dehydrogenase according to claim 1 and an electron transfersubunit.
 6. A glucose assay kit comprising the mutant glucosedehydrogenase according to claim 1, optionally in combination with anelectron transfer subunit.
 7. A glucose sensor comprising the mutantglucose dehydrogenase according to claim 1, optionally in combinationwith an electron transfer subunit.
 8. A mutant glucose dehydrogenasecomplex comprising at least the isolated mutant glucose dehydrogenaseaccording to claim 4 and an electron transfer subunit.
 9. A glucoseassay kit comprising the mutant glucose dehydrogenase according to claim4, optionally in combination with an electron transfer subunit.
 10. Aglucose sensor comprising the mutant glucose dehydrogenase according toclaim 4, optionally in combination with an electron transfer subunit.11. A mutant glucose dehydrogenase complex comprising at least theisolated mutant glucose dehydrogenase according to claim 2 and anelectron transfer subunit.
 12. A glucose assay kit or glucose sensorcomprising the mutant glucose dehydrogenase according to claim 2,optionally in combination with an electron transfer subunit.
 13. Amutant glucose dehydrogenase complex comprising at least the isolatedmutant glucose dehydrogenase according to claim 3 and an electrontransfer subunit.
 14. A glucose assay kit or glucose sensor comprisingthe mutant glucose dehydrogenase according to claim 3, optionally incombination with an electron transfer subunit.